Cells possess several types of volume-sensitive anion channels that play specific roles in the recovery from swelling. We have shown that the swelling-activated, outwardly rectifying anion channel functions as the major pathway for volume regulatory loss of organic osmolytes. We have termed this channel VSOAC for Volume-Sensitive Organic osmolyte/Anion Channel. This proposal has two broad goals aimed at defining the cellular and molecular physiology of VSOAC. First, we will test the hypothesis that the protein ICln is VSOAC or a functional channel subunit. ICln has ben proposed to be the outwardly rectifying anion channel or a channel regulator. Based on our recent studies, we propose that ICln is the VSOAC channel, that the channel is porin-like' in nature, and that channel activation occurs by insertion of the protein into the plasma membrane. This hypothesis is supported strongly by our demonstration that recombinant ICln protein reconstituted into planar lipid bilayers functions as a rectifying, anion-selective channel with the properties of VSOAC. Our studies will characterize the reconstituted channel using electrophysiological, mutagenesis and isotope flux methods. We will also use biochemical, molecular, immunolocalization and electrophysiological strategies to determine if LClin is inserted into the plasma membrane. Our second goal is to characterize the volume-sensitive anion channels that function in concert with VSOAC allowing cells to sense and respond selectively to different types of volume perturbations. We propose that VSOAC is activated only when cell Cl is insufficient to mediate volume recovery, that more Cl-selective anion channels are activated for volume regulatory electrolyte loss, and that intracellular CI" levels coordinate the activity of different types of anion channels. This hypothesis is supported strongly by our observation that VSOAC in skate hepatocytes and rat C6 glioma cells is inhibited by increased intracellular Cl" and electrolyte levels. Despite complete inhibition of VSOAC,m however, normal volume recovery occurs in response to swelling. This volume recovery is mediated by an anion channel that is impermeable to organic osmolytes. We will use patch clamp methods to characterize the basic electrophysiological properties of this channel and to assess whether its activity is modulated by intracellular Cl" concentration.